This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ABSTRACT The outcome for patients with recurrent/metastatic lung cancer has not changed over the last 2 decades and most patients die of their disease. New anti-lung cancer therapies are needed and immunotherapy holds the promise to fulfill this need. The bodys immune defense against cancers often fails because cancers either do not induce or actively inhibit immunity. We will counteract these limitations by i) engineering killer T cells or recognize structures on lung cancer cells and ii) to resist the defenses imposed by the cancer cell environment. Using gene transfer technology, it may be possible to take advantage of the tumor killing ability of killer T cells and render them resistant to inhibitory factors secreted by tumor cells. We propose taking the patients own T cells and putting into them two genes. The first gene is for an artificial structure (receptor) that will direct the T cells to the tumor and the second gene is for a dominant negative receptor (DNR), which renders T cells resistant against one of the major inhibitory factors secreted by lung cancer cells. We will place the two genes into T cells that are pre-selected for their ability to recognize the Epstein Barr virus which is chronically present in most people. Because these EBV-specific T cells meet the virus positive cells as well as tumor cells, they receive extra stimulation. We will put the genes for the artificial receptor and the DNR into patients EBV-specific T cells using a modified virus (Moloney retroviral vector). These modified T-cells will then be given directly into the patients blood stream through a central line or a vein. Patients will be treated in the clinic and will be monitored closely for several hours after infusion. We will collect samples of blood from peripheral blood at regular intervals. We will look for the safety, the persistence and the function of the cells we put into the patients. Ultimately we hope to get evidence that these modified T cells are effective at fighting the cancer. I. HYPOTHESIS From our experience, the persistence of infused EBV-CTL in cancer patients is limited, most likely due to immune evasion strategies adapted by cancers. For example, lung cancers express high levels of TGF, a potent negative regulator of immune cells. In preclinical studies we have shown that EBV-CTL can be made resistant to the inhibitory effects of TGF by expressing a dominant negative TGF receptor II (DNR). DNR expressing CTL retained their effector function and had enhanced antitumor activity in preclinical animal models. Importantly, DNR expressing CTL did not show any evidence of autonomous (antigen-independent) CTL proliferation. These results provided the rationale for a RAC and FDA approved clinical trial (RAC #0507-724), in which we are testing the safety and efficacy of (TGF)-resistant CTL in lymphoma patients. In this study we will generate autologous EBV-specific CTL and genetically modify them using a retroviral vector expressing a dominant negative TGF beta receptor II (DNRII) to render them resistant to the immunosuppressive effects of TGF beta, and a chimeric antigen receptor recognizing HER2. Two separate vectors will be used to transfer the genes. Each cell product will thus contain: 1) Unmodified CTLs 2) HER2 CAR CTLs 3) TGF[unreadable]] DNR CTLs 4) HER2 CAR and TGF beta DNR CTLs We will adoptively transfer these CTLs to patients with advanced HER2-positive lung cancer. We will assess the safety of the infusion and measure the survival of the gene modified cells in subgroups 2, 3 and 4. We will detect each cell population by quantitative PCR and by immunostaining of peripheral blood and in tumor tissue/other fluids (when available). We anticipate that the CTL populations expressing TGF[unreadable] DNR (subgroups 3 and 4) will survive better than the population lacking this modification (subgroup 2). We also anticipate survival of groups 3 and 4 will be equivalent, but if CAR expression itself has an adverse influence on CTL survival, then subgroup 3 will come to dominate. Alternatively, if cells expressing both TGF[unreadable] DNR and the CAR localize better in tumors, then we would expect to see dominance of subgroup 4 in tissue samples. II. SPECIFIC AIMS Primary: To determine the safety of one intravenous injections of autologous Transforming Growth Factor (TGF)-resistant cytotoxic T lymphocytes (CTLs) directed to Epstein Barr virus (EBV) through their native receptor and HER2 through their chimeric antigen receptor (CAR) in patients with advanced HER2-positive lung cancers. Secondary: To compare the survival and the immune function of the TGF-resistant and non resistant components of the infused CAR-CTL. To assess the anti-tumor effects of the infused CAR-CTL.